Figure 2. Assembly-dependent trafficking in merozoites and a role in host cell invasion for RhopH3 alone.
(A) Strategy for the introduction of the PA epitope tag followed by a stop codon and a glmS riboswitch at the 3’ end of rhoph genes. (B) Schematic showing GlcN exposure timed to cover the expression of rhoph genes and the subsequent harvest of cells for immunoblotting (B), indirect immunofluorescence assays (IFA), and invasion studies (Inv). (C) Immunoblots of matched lysates from schizont-stage parasites with or without exposure to 4 mM GlcN. Knockdown eliminates targeted gene product and also CLAG3. RAP1, an unassociated rhoptry protein, is preserved. (D) IFA of indicated proteins in R2glmS and R3glmS at schizont stage; the colocalization of a protein with RAP1 and apical puncta in daughter merozoites indicates rhoptry trafficking. Scale bars, 5 µm. (E) GlcN dose responses (mean ± S.E.M.) for merozoite invasion by each parasite. (F) Giemsa-stained micrographs of R3glmS showing extracellular merozoites (red arrows) after GlcN treatment, but not in the untreated control. Immature ring-infected erythrocytes are also apparent.